1. Field of the Invention
The invention relates to the application of sequestrants and inhibitors of protease to wounds for the purpose of enhanced healing. The invention also relates to the inclusion of these active agents in wound dressings to which they may be,either compositioned or bound by covalent or ionic means for the purpose of controlled release or sequestration.
2. Description of the Prior Art
A wound of the skin is any degradation of its normal structure and function resulting from an internal or external pathology. A healing wound has aspects relating to control of infection, resolution of inflammation, angiogenesis, regeneration of a functional connective tissue matrix, contraction, resurfacing, differentiation, and remodeling. Chronic wounds are wounds that don""t heal in a timely process.
Chronic wounds represent a worldwide health problem. Chronic wounds are a growing health care problem largely due to increasing longevity of the American population. Pressure or decubitus ulcers which are a type of chronic wound represent an estimated 3% to 5% incidence in hospital patients. In patients with spinal chord injuries the incidence of chronic wounds is 25% to 85%. Approximately one million Americans hospitalized yearly will develop pressure sores resulting in a cost of 750 million dollars for patient care.
Elastase is perhaps the most destructive enzyme in the body and has been well characterized in non-healing wounds. An excessive concentration of both the serine protease elastase and matrix metalloproteinases (MMPs) in chronic non-healing wounds has been shown to deleteriously degrade cytokine growth factors, fibronectin, and endogenous levels of protease inhibitors necessary for healing. Although numerous studies with both animals and human beings have shown that growth factors may accelerate healing of chronic wounds, therapeutic attempts to modulate the wound healing response with them have had limited success.
The composition of the wound dressing is relevant to designing a mechanism-based approach to protease inhibition in the environment of the wound fluid. (Wiseman D M, Rovee, D T, Alvarez O M Wound dressing: design and use in Wound Healing Biochemical and Clinical Aspects, eds. Cohen I K, Diegelmann, R F, Lindbald, W J, 1992, Hartcourt Brace Jovanovich, Inc. 562-580). The fiber or gel composition of synthetic dressings, applied to chronic wounds, include synthetic hydrogel polymers, collagen, hydrocolloids, alginates and cotton and carboxymethylcellulose. Controlled release of agents linked with important roles in wound healing includes growth factors, antibiotics, and trace elements. The use of the enzyme inhibitor aprotinin for treatment of corneal ulcers was reported, however, there have been no known reports of treatment methods on the release of elastase inhibitors into wounds.
U.S. Pat. No. 5,098,417 to Yamazaki et al. teaches the ionic bonding of physiologically active agents to cellulosic wound dressings.
U.S. Pat. No. 4,453,939 to Zimmerman et al. teaches the inclusion of aprotonin in compositions for xe2x80x9csealing and healingxe2x80x9d of wounds.
U.S. Pat. No. 5,807,555 to Bonte et al. teaches the inclusion of inhibitors for alpha-1-protease, collagenase, and elastase in pharmaceutical compositions for promotion of collagen synthesis.
U.S. Pat. No. 5,696,101 to Wu et al. teaches use of oxidized cellulose (e.g. oxycel) as a bactericide and hemostat in treatment of wounds.
World Patent WO 98/00180 to Watt et al. teaches complexation of oxidized cellulose with structural proteins (e.g. collagen) for chronic wound healing; and references the utility of oligosaccharide fragments produced by the breakdown of oxidized cellulose in vivo in the promotion of wound healing.
We have now discovered that direct application of protease inhibitors to wound sites promotes wound healing by amelioration of the deleterious effects of elastase on cytokines, endogenous anti-proteases, and fibronectin in the wound microenvironment. While not wanting to be bound thereto, it is Applicants"" theory that the release of protease inhibitors into the chronic wound or modification of wound dressing fibers for selective protease sequestration may be beneficial in restoring the proteinase/antiproteinase balance needed to avoid degradation of growth factors and effectively accelerate healing of chronic wounds. The active agents are applied to the wound site in conjunction with known wound dressings, preferably polysaccharide based wound dressings. The invention includes methods of linking these protease inhibitors either covalently or ionically to wound dressings including polysaccharide containing matrices. Also described are formulations of the inhibitors onto fibers, the method of production of same, and pharmaceutical compositions which are effective in the treatment of mammalian chronic wounds.
Therefore, it is an object of this invention to provide methods and compositions for the enhanced treatment of mammalian wounds comprising the application of protease inhibitors.
Another object is to provide novel conjugates of protease inhibitors with wound dressings for use as sequestrants in wound environments.
A further object is to provide ionic systems for sustained release of protease inhibitors into the wound environment.
Other objects and advantages will become apparent from the ensuing description.
The present invention is based upon the previously unrecognized discovery that inhibitors and sequestrants of proteases may be used as healing accelerants of chronic wounds. These may be physically applied on wound dressings, or in the alternative may be ionically or covalently conjugated to a wound dressing material for purposes of sustained release of active agent or sequestration of endogenous constituents from the wound environment. The term protease inhibitor is meant to include those materials which effect a diminution in protease activity in the wound environment.
Specific pharmacological effects of elastase inhibitors and sequences associated with wound dressings include inhibition of the breakdown of growth factors that stimulate migration of cells to the ulcer site of the wound leading to the growth of new tissue that heals the open wound. This technology is broadly applicable to all forms of chronic wounds including diabetic ulcers and decubitus bedsores. Both peripheral and central administration of the compounds formulated on wound dressings accelerate wound healing of chronic wounds. The compounds of this invention may be applied to wound dressings as agents that may be released into the wound and thereby inhibit human elastase and thus prevent growth factor and tissue degradation. Alternatively the inhibitors of this invention are covalently bound to the wound dressing. As a component of such a matrix, they are able to sequester destructive elastase from the microenvironment of the wound, thus preventing the degradation of growth factors and fibronectin that would otherwise occur.
The therapeutic administration of the modified wound dressings containing inhibitors include a pharmacologically effective dose of the inhibitor or sequestrant when used in the treatment of a patient in need thereof. The dose of inhibitor or sequestrant required on the wound dressing to promote accelerated healing in the patient ranges from about 0.2 mg/gram fiber to about 200 mg/gram fiber per day, with this in turn being dependent upon specific factors including patient health, wound type, and specific-protease inhibitor/sequestrant utilized. The amount of active agent required can be readily determined by those skilled in the art.
The term patient used herein is taken to mean mammals such as sheep, horses, cattle, pigs, dogs, cats, rats, mice and primates, including humans.
The term wound dressing used herein is taken to include any pharmaceutically acceptable wound covering such as:
a) films, including those of a semipermeable or a semi-occlusive nature such as polyurethane copolymers, acrylamides, acrylates, paraffin, polysaccharides, cellophane and lanolin.
b) hydrocolloids including carboxymethylcellulose, protein constituents of gelatin, pectin, and complex polysaccharides including Acacia gum, guar gum and karaya.
xe2x80x83These materials may be utilized in the form of a flexible foam or, in the alternative, formulated in polyurethane or, in a further alternative, formulated as an adhesive mass such as polyisobutylene.
c) hydrogels such as agar, starch or propylene glycol; which typically contain about 80% to about 90% water and are conventionally formulated as sheets, powders, pastes and gels in conjunction with cross-linked polymers such as polyethylene oxide, polyvinyl pyrollidone, acrylamide, propylene glycol.
d) foams such as polysaccharide analogs which consist of a hydrophilic open-celled contact surface and hydrophobic closed-cell polyurethane
e) impregnates including pine mesh gauze, paraffin and lanolin-coated gauze, polyethylene glycol-coated gauze, knitted viscose, rayon, and polyester.
f) cellulose-like polysaccharides such as alginates, including calcium alginate, which may be formulated as non-woven composites of fibers or spun into woven composites.
Preferred wound dressings are polysaccharide-containing matrices capable of ionically or covalently bonding the active agents thereto, or having the active agent compositioned with or upon, and is envisioned to include chitosans, alginates and cotton or carboxymethylated cotton in the form of gauze, films, hydrocolloids, hydrogels, hydroactives, foams, impregnates, absorptive powders and pastes, as known in the art and described in USP 24:NF 19; The United States Pharmacopeia: The National Formulary, USP 24:NF 19, United States Pharmacopeial Convention, INC., Rockville, Md., Jan. 1, 2000, incorporated by reference herein.
Especially preferred wound dressings include cotton cellulose formed as woven or non-woven gauze wherein the protease sequestrant or inhibitor is linked to the cellulose polysaccharide chain through a chemical sustituent selected from the group consisting of amino, carboxylate, citrate, phosphate, sulfonate, chloride, bromide, mono-carboxylic acid, di-carboxylic acid, tri- carboxylic acid; or any pharmaceutically acceptable salt thereof. Exemplary salts are seen to include those of acids such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, and 2-phenoxybenzoic; and sulfonic acids such as methane sulfonic acid and hydroxyethane sulfonic acid. Salts of the carboxy terminal amino acid moiety may include the nontoxic carboxylic acid salts formed with any suitable inorganic or organic bases. Illustratively, these salts include those of alkali metals, as for example, sodium and potassium; alkaline earth metals, such as calcium and magnesium; light metals of Group IIA elements including aluminum; and organic primary, secondary, and tertiary amines, as for example, trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine, N,Nxe2x80x2-dibenzylethylenediamine, dihydroabietylamine, N-alkylpiperidine and any other suitable amine.
The active agents may be applied as a reactively bound constituent of a wound dressing or may be compositioned for application to a treatment site via moistened fibers in the dressing. Dressing systems may be either single or multi-phase; with the one-phase system consisting of the wound dressing with the active agent. An exemplary multi-phase system would employ the wound dressing and a suspension of a physiologically acceptable diluent. Exemplary pharmaceutical carriers which may function as the diluent can be a sterile physiologically acceptable liquids such as water and oils and may optionally further contain surfactants and other pharmaceutically acceptable adjuvants. An exemplary but non-exhaustive list of oils which can be employed in these preparations are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general water, saline, and glycols, such as polyethylene glycols are preferred liquid carriers.
The present invention is thus viewed to encompass derivatives of wound dressings presented as formulas I and II below:
Xxe2x80x94Axe2x80x83xe2x80x83(I)
wherein:
X is selected from:
a) films, including those of a semipermeable or a semi-occlusive nature such as polyurethane copolymers, acrylamides, acrylates, paraffin, polysaccharides, cellophane and lanolin;
b) hydrocolloids including carboxymethylcellulose, protein constituents of gelatin, pectin, and complex polysaccharides including Acacia gum, guar gum and karaya.
xe2x80x83These materials may be utilized in the form of a flexible foam or, in the alternative, formulated in polyurethane or, in a further alternative, formulated as an adhesive mass such as polyisobutylene;
c) hydrogels such as agar, starch or propylene glycol; which typically contain about 80% to about 90% water and are conventionally formulated as sheets, powders, pastes and gels in conjunction with cross-linked polymers such as polyethylene oxide, polyvinyl pyrollidone, acrylamide, propylene glycol;
d) foams such as polysaccharide analogs which consist of a hydrophilic open-celled contact surface and hydrophobic closed-cell polyurethane;
e) impregnates including pine mesh gauze, paraffin and lanolin-coated gauze, polyethylene glycol-coated gauze, knitted viscose, rayon, and polyester;
f) cellulose-like polysaccharides such as alginates, including calcium alginate, which may be formulated as non-woven composites of fibers or spun into woven composites;
g) polysaccharide-containing matrices capable of ionically or covalently bonding the active agents thereto, or having the active agent compositioned with or upon, and is envisioned to include chitosans, alginates and-cotton or carboxymethylated cotton in the form of gauze, films, hydrocolloids, hydrogels, hydroactives, foams, impregnates, absorptive powders and pastes; and
h) cotton cellulose formed as woven or non-woven gauze wherein the protease sequestrant or inhibitor is linked to the cellulose polysaccharide chain through a chemical sustituent selected from the group consisting of amino, carboxylate, citrate, phosphate, sulfonate, chloride, bromide, mono-carboxylic acid, di-carboxylic acid, tri-carboxylic acid; or any pharmaceutically acceptable salt thereof.
xe2x80x83Exemplary salts are seen to include those of acids such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, and 2-phenoxybenzoic; and sulfonic acids such as methane sulfonic acid and hydroxyethane sulfonic acid. Salts of the carboxy terminal amino acid moiety may include the nontoxic carboxylic acid salts formed with any suitable inorganic or organic bases.
xe2x80x83Illustratively, these salts include those of alkali metals, as for example, sodium and potassium; alkaline earth metals, such as calcium and magnesium; light metals of Group IIA elements including aluminum; and organic primary, secondary, and tertiary amines, as for example, trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine, N,Nxe2x80x2-dibenzylethylenediamine, dihydroabietylamine, -alkylpiperidine and any other suitable amine.
A is selected from the group consisting of hydrogen, methyl, methyl chloroformate, pentafluoroethylketone, dihydroxylketone, levulinate, alkyl ketone, 2,3 dialdehyde anhydroglucose, glucose, glucose-6-citrate, 2,3 dialdehydo-glucose-6-citrate, an alkyl amino acid such as Ala, Leu, Ile, Val, and Nle, or a di- or tri-peptide sequence consisting of the following sequence: Val-Pro, Pro-Val, Ala-Pro-Val or Val-Pro-Ala or a tetrapeptide sequence wherein any of the following amino acids are the amino terminal residue of Ala-Pro-Val or Val-Pro-Ala; Lys, Arg, Trp, Phe, Gin, His, Tyr, and are linked through the amino- or COOH-termini of the peptide to X; with the proviso that when A1 is an amino acid or peptide it may also be derivatized at its unbound amino- or COOH-terminus as an acid, carboxamide, alcohol, ester, ketone, aldehyde, ketomethylester, methyl chloroformate, pentafluoroethylketone or p-nitroanilide.
Compounds of formula II are of the structure:
Xxe2x80x94Bxe2x80x83xe2x80x83(II)
wherein:
X is selected from:
a) films, including those of a semipermeable or a semi-occlusive nature such as polyurethane copolymers, acrylamides, acrylates, paraffin, polysaccharides, cellophane and lanolin;
b) hydrocolloids including carboxymethylcellulose, protein constituents of gelatin, pectin, and complex polysaccharides including Acacia gum, guar gum and karaya.
xe2x80x83These materials may be utilized in the form of a flexible foam or, in the alternative, formulated in polyurethane or, in a further alternative, formulated as an adhesive mass such as polyisobutylene;
c) hydrogels such as agar, starch or propylene glycol; which typically contain about 80% to about 90% water and are conventionally formulated as sheets, powders, pastes and gels in conjunction with cross-linked polymers such as polyethylene oxide, polyvinyl pyrollidone, acrylamide, propylene glycol;
d) foams such as polysaccharide analogs which consist of a hydrophilic open-celled contact surface and hydrophobic closed-cell polyurethane;
e) impregnates including pine mesh gauze, paraffin and lanolin-coated gauze, polyethylene glycol-coated gauze, knitted viscose, rayon, and polyester;
f) cellulose-like polysaccharides such as alginates, including calcium alginate, which may be formulated as non-woven composites of fibers or spun into woven composites;
g) polysaccharide-containing matrices capable of ionically or covalently bonding the active agents thereto, or having the active agent compositioned with or upon, and is envisioned to include chitosans, alginates and cotton or carboxymethylated cotton in the form of gauze, films, hydrocolloids, hydrogels, hydroactives, foams, impregnates, absorptive powders and pastes; and
h) cotton cellulose formed as woven or non-woven gauze wherein the protease sequestrant or inhibitor is linked to the cellulose polysaccharide chain through a chemical sustituent selected from the group consisting of amino, carboxylate, citrate, phosphate, sulfonate, chloride, bromide, mono-carboxylic acid, di-carboxylic acid, tri-carboxylic acid; or any pharmaceutically acceptable salt thereof.
xe2x80x83Exemplary salts are seen to include those of acids such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, and 2-phenoxybenzoic; and sulfonic acids such as methane sulfonic acid and hydroxyethane sulfonic acid. Salts of the carboxy terminal amino acid moiety may include the nontoxic carboxylic acid salts formed with any suitable inorganic or organic bases.
xe2x80x83Illustratively, these salts include those of alkali metals, as for example, sodium and potassium; alkaline earth metals, such as calcium and magnesium; light metals of Group IIA elements including aluminum; and organic primary, secondary, and tertiary amines, as for example, trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine, N,Nxe2x80x2-dibenzylethylenediamine, dihydroabietylamine, N-alkylpiperidine and any other suitable amine.
B is selected from the group consisting of an alkyl amino acid such as Ala, Leu, Ile, Val, and Nle; a di- or tri-peptide sequence consisting of Val-Pro, Pro-Val, Ala-Pro-Val or Val-Pro-Ala; or a tetrapeptide sequence containing Ala-Pro-Val or Val-Pro-possessing as a terminal residue amino acids selected from Ala, Lys, Arg, Trp, Phe, Gln, His, and Tyr; and wherein the substituent is linked, through the amino- or COOH-termini of the peptide, to X via a salt bridge, or are embedded in the cotton cellulose fiber; with the proviso that when A1 is an amino acid or peptide, it may also be derivatized at its unbound amino- or COOH-terminus as an acid, carboxamide, alcohol, ester, ketone, aldehyde, ketomethylester, methyl chloroformate, pentafluoroethylketone, or p-nitroanilide. B1 may also be alpha-antitrypsin or any protein serine protease inhibitor bound through a salt bridge to the derivatized cotton cellulose. In a similar manner B1 may be an antibiotic such as doxycycline or cephalosporin linked via a salt bridge or embedded in the cotton fiber.